Interpretive Summary: An important but poorly understood step in the regulation of hormone secretion is the sorting of hormones during intracellular synthesis into either the constitutive secretory pathway, in which they are secreted constantly, or the regulated secretory pathway, in which they are packaged into secretory granules and released into the circulation in response to chemical or neural signals. Recent work in mammals has suggested that an intracellular protein, chromogranin A (CgA), has primary responsibility for this cellular regulation step. In this research, we produced a monoclonal antibody to turkey CgA and studied the changes in CgA protein expression in different pituitary cell types in both young and adult turkeys and chickens. We also purified turkey CgA using our antibody and confirmed its identity proteomic amino acid sequencing techniques. We used public nucleotide databases to clone the chicken CgA complementary DNA sequence and compared the translated amino acid sequence of chicken CgA to our experimentally-determined turkey CgA sequence. Our results showed that turkey and chicken CgA are found in all pituitary cells that contain luteinizing hormone in the immature bird, but CgA is absent in these cells in the adult laying bird. Conversely, CgA is absent in pituitary cells which produce growth hormone in the growing bird, but is abundant in some, but not all, such cells in the adult. This is the first demonstration in any species of: 1) an age or reproduction-related change in CgA production in pituitary cells; and 2) the presence of CgA in growth-hormone producing cells. This work is important because it suggests that changes in CgA synthesis may provide an important regulatory pathway for controlling the secretion of luteinizing hormone and growth hormone. The information will be used by scientists in planning future research, and the monoclonal antibodies produced in these studies will be provided to the research community for further studies.

Technical Abstract:
In the course of producing monoclonal antibodies to turkey prolactin, three monoclonal antibodies to turkey chromogranin A (CgA) were also produced, apparently arising from minor contamination of the turkey prolactin immunogen with peptide fragments of CgA. The identity of the antigen recognized by these antibodies was established by tandem mass spectrometry de novo sequencing of seven tryptic peptides from a turkey pituitary protein purified by immunoaffinity chromatography. These peptides showed high homology with distinctly separate regions of mammalian and ostrich CgA, and in silico cloned chicken CgA sequences. Chromogranin A immunostaining patterns on Western blots and pituitary tissue sections differed from those of prolactin, growth hormone, or luteinizing hormone (LH). Dual-label fluorescent immunohistochemistry revealed that CgA was co-localized with LH in most avian gonadotrophs in young chickens and turkeys, but not in adult, laying birds. Conversely, CgA was found in a majority of somatotrophs in laying birds but was absent from somatotrophs in young, growing chickens and turkeys. Lactotrophs contained no detectable CgA immunoreactivity in the tissues studied. These results suggest that CgA may modulate hormone secretion by gonadotrophs and somatotrophs in a manner that differs between cell type with age or reproductive state.